Ultrastructural aspects of the migration of somite cells to the posterior limb buds of mice]

Migrating cells originating selectively in the ventral lateral edge of the somites adjacent to the mouse hindlimb bud have been studied in transverse sections by transmission and scanning electron microscopy. Collected during the 10th and 11th gestational days, the embryos have been classified according to the number of metameres. As soon as the 28 somite stage, discrete cytological modifications occur in a limited caudal area of the ventro-lateral somitic edge. Loosing the typical epithelial arrangement characteristic of the dermatome cells, these ventral cells show large areas of close contact between their plasma membrane and a superficial microfilamentous material accumulates in the contact areas. At the 33 somite stage, the same groups of cells elongate and form long cellular trails invading the proximal area of the limb bud mesoderm. The migrating cells become polarized along the migrating axis and they retain large and smooth intercellular contacts with each other. Very selective ultrastructural features of the migrating somitic cells can be interpreted in relation to their cinetic activity or to their early myogenic differentiation. In addition to their mutual superficial relationships, the migrating cells are characterized by the presence of numerous oriented microtubules, of a high number of active mitochondria, of an abundant granular endoplasmic reticulum and of an hypertrophied Golgi apparatus regularly located near the nucleus in the "trailing" edge of the cells. Several dense granules with a diameter of 8 nm are present in the mitochondrial matrix. The extensive Golgi apparatus is associated to numerous thick walled vesicles, which increase from 60 to 150 nm in diameter as they become closer and closer to the plasma membrane. These vesicles are absent in the mesodermal cells of somatopleural origin; their presence in the migrating somitic cells is probably related to an early myogenic differentiation. The observation made near the distal end of the somitic cellular trails suggests that the more distal somitic cells rapidly loose their ultrastructural particularities as soon as they are dispersed in the limb bud mesoderm; this aspect of the processus, however, requires the study of later developmental stages. Other observations made in the same material bring some precisions to the ecto-mesodermal relationships which are established in the apical area of the limb bud. Scanning electron microscopic observations of thick sections reveal that the outer mesodermal cells of this area send numerous filopodia which make contact with the basement membrane underlying the apical ectodermal ridge.     

Differential cell affinity and sorting of anterior and posterior cells during outgrowth of recombinant avian limb buds

To examine the role of position-specific differences in cell-cell affinity, recombinant limb buds composed of dissociated and reaggregated cells derived from anterior (A) and posterior (P) limb bud fragments were analyzed. Dissociated anterior and/or posterior cells were differentially labeled, and their behavior was analyzed during recombinant limb bud outgrowth. We find that anterior and posterior cells sort out from one another to form alternating anterior and posterior stripes of cells that extend distally along the proximal-distal axis. These alternating stripes are prominent across the A/P axis in whole-mount preparations of recombinant limb buds after 48 h of outgrowth when the presumptive autopod is dorsal-ventrally flattened and digit rudiments are not evident. After 96 h, when digital and interdigital regions are clearly defined, we find evidence that A/P stripes do not follow obvious anatomical boundaries. The formation of A/P stripes is not inhibited by grafts of ZPA tissue, suggesting that polarizing activity does not influence cell-cell affinity early in limb outgrowth. In vitro studies provide evidence that cell sorting is not dependent on the limb bud ectoderm or the AER; however, cells sort out without organizing into stripes. Gene expression studies using anterior-specific (Alx-4) and posterior-specific (Shh, Bmp-2, and Hoxd-13) marker genes failed to reveal expression domains that corresponded to stripe formation. Control recombinant limb buds composed of anterior, central, or posterior mesenchyme formed digits in a position-specific manner. A/P recombinant limb buds that develop to later stages form digits that are characteristic of central recombinant limbs. These data provide the first definitive evidence of A/P cell sorting during limb outgrowth in vivo and suggest that differential cell affinities play a role in modulating cell behavior during distal outgrowth.     

Feather buds exert a polarizing activity when transplanted to chick limb buds

Homeoproteins have been shown to be expressed in a position-specific manner along the anterior-posterior axis in the developing chick feather bud, as seen also in the developing limb bud. These facts raise the possibility that there may be common mechanistic features in the establishment of the anterior-posterior polarity between both organs. In order to investigate this possibility, feather bud tissues were transplanted into the anterior region of limb buds to determine whether feather bud tissues possess properties such as the zone of polarizing activity of the limb bud. The manipulated limb bud formed a mirror image duplication of the skeletal elements, mainly (2)2234 digit pattern or sometimes 3(2)234. Both the anterior and posterior halves of feather bud tissue exhibited almost equal activity in inducing ectopic skeletal elements. Hox d-12 and Hox a-13 were expressed coordinately around the transplanted site of the operated limb bud. This secondary axis-inducing activity of the feather bud was enhanced when grafts were pretreated with trypsin. In contrast, the presumptive feather bud tissue and inter-feather bud tissue did not induce a secondary axis of the limb bud. These results suggest that the feather bud contains a region that exerts polarizing activity and that this region may play key roles in the formation of the anterior-posterior and, if it exists, proximal-distal axis of the feather bud, possibly via the regulation of region specific expression of Hox genes.     

Myocyte-specific enhancer factor 2A is essential for zebrafish posterior somite development

Somite development is governed tightly by genetic factors. In the large-scale mutagenesis screens of zebrafish, no mutations were linked to myocyte enhancer factor 2A (MEF2A) locus. In this study, we find that MEF2A knock-down embryos display a downward tail curvature and have U-shaped posterior somites. Furthermore, we demonstrate that MEF2A is required for Hedgehog signaling. MEF2A inhibition results in induction of apoptosis in the posterior somites. We further find that Hedgehog signaling can negatively regulate MEF2A expression in the somites. Microarray studies reveal a number of genes that are differentially expressed in the MEF2A morphants. Our studies suggest that MEF2A is essential for zebrafish posterior somite development.     

Mammalian limb bud development: in situ fate maps of early hindlimb buds

Fate maps of the developing mouse hindlimb bud have been constructed for the first time using exo utero surgical techniques and carbon particle injections. Such fate maps demonstrate that the limb develops in a proximal to distal manner as a result of distal expansion. The anterior-posterior extent of the limb bud develops asymmetrically with the posterior half giving rise to slightly more of the digit pattern (digits 3-5) than the anterior half (digits 1 and 2). We found no evidence for the occurrence of extensive cellular rearrangements during limb development, and the free limb bud appears to give rise to only zeugo- and autopodial elements with the stylopod arising in the body wall proximal to the bud. These results are consistent with our current understanding of limb development in lower vertebrates and also provide detailed information that will be useful for future limb studies in mammals.     

Analysis of normal somite development

We describe how the first 6 somite pairs form, using the third somites as examples. This history is based upon time-lapse movies of carbon-marked embryos and histological studies by light and electron microscopy of embryos fixed in situ with glutaraldehyde and osmium tetroxide. At head-process stage a continuous sheet of mesoblast occupies the regions of the future third somites. Mesoblast cells attach either to hypoblast or to overlying neural plate which is already a simple pseudostratified columnar epithelium. Prospective somite cells are those attached to the neuroepithelium, and they extend laterally exactly as far as the neural plate does. By head-fold stage, regression of the node down the midline is shearing the sheet of mesoblast into right and left halves. Somite cells hang from the bottom of the neural plate. As the neural plate condenses toward the midline, attached somite cells are compacted. When the somite segments, somite cells are tightly apposed to one another, and, in addition to junctions binding their basal ends, new junctions appear between their apical ends. This leads to reorganization into the typical somite rosette configuration. Spaces filled with extracellular materials form around the whole somite.     

Growth and differentiation of fetal rat limb buds transplanted in athymic (nude) mice

Limb buds of day 14 rat fetuses were cut into pieces and transplanted into the subcutaneous tissue of athymic (nude) mice. In day 14 fetal limbs, mesenchymal cells have begun to condense to form cartilaginous anlage, but no cartilage has been formed. Within 7 days after grafting, masses of hyaline cartilage developed. Numerous osteoblasts appeared, and new bone formation began by 14 days. By 20 days, osteoclasts appeared, and the formation of bone trabeculae and marrow cavities progressed. The cytological characteristics of chondrocytes, osteoblasts and osteoclasts were essentially the same as those seen in vivo. Many grafts developed into long bones, having the diaphysis and epiphysis. The mode of chondrogenesis and osteogenesis in the grafts was histologically similar to the corresponding process in vivo, although the differentiation was slower in the grafted limbs. Since the grafted limb buds showed remarkable growth and tissue differentiation for at least several weeks, this heterotransplantation system would be of potential use for the study of bone formation and resorption as well as for developmental toxicological studies.     

Congenic method in the chick limb buds by electroporation

Electroporation is a powerful tool with which to study limb development. Limb development, however, remains an intricate series of events, requiring the precise dissection of developmental processes using relevant transgenes. In this review, we describe the anatomy of the limb field as the basis of targeted electroporation, and specific expression vectors are discussed. We share a useful protocol for electroporation of chick limb buds, and the expression pattern of enhanced green fluorescent protein in the limb buds is used to demonstrate relevant embryonic patterning. Finally, useful trouble-shooting techniques are described.     

Evidence for polarizing zone in the limb buds of Xenopus laevis

To test for the presence of polarizing mesoderm in an amphibian, Xenopus laevis hindlimb bud tips were rotated 180° on the proximodistal axis and returned to the stump. Supernumerary outgrowths were induced in the preaxial stump and preaxial tip tissues, and the most postaxial digit always formed next to the grafted postaxial tissue. The occurrence of polarized supernumerary outgrowths indicated that the posterior limb border contained a polarizing zone. When the limb tip was cut at varying known lengths from the body wall, rotated, and grafted to the limb stump, the incidence of twinning along the proximodistal axis permitted insight into the distribution of the polarizing zone along the posterior border. The location of polarizing tissues was found to be similar to that in the chick wing bud at comparable stages. To confirm the posterior border stump influence on the rotated preaxial limb tip tissues, 180° tip rotations were made at the proximodistal level with the highest incidence of twinning. In these cases, the adjacent stump posterior border tissues (polarizing zone) were removed, leaving a substantial amount of the deeper postaxial stump tissue, however. The frequency of twinning from tip tissues was greatly reduced in these larvae compared to those with rotated limb tips on intact stumps. Cytological examination of supernumerary outgrowths resulting from grafts of two-nucleolate tips onto one-nucleolate stumps confirmed the preaxial source of the supernumerary outgrowths.     

Abnormalities of somite development in the absence of retinoic acid

We describe the effects of an absence of retinoic acid (RA) on the development of somites in the quail embryo. RA was removed by generating vitamin A deficient quail embryos whereupon the resulting defects in the embryos can be analysed. The effect on the somites is threefold. Firstly, they are half the size of normal, but the total number of somites is the same as normal. There has therefore been some global regulation event. Secondly, by TUNEL staining and TEM we show that the lateral halves of all of the somites undergo apoptosis between stages 11 and 14. This effect is confined to the sclerotome of the somites. Thirdly, some of the genes involved in somite differentiation are down-regulated such as fgf-4, fgf-8, engrailed and myogenin whereas others we examined such as cek-8, Delta, follistatin and myf5 are not affected. These studies reveal remarkably specific effects of RA on developmental gene pathways in the embryo.